This invention relates generally to mass spectrometry, and in particular to matrix assisted laser desorption and ionization time-of-flight mass spectrometry. Specifically, this invention relates to a method and apparatus for improved sample desorption by laser excitation that results in greatly enhanced mass resolution and sensitivity for a time-of-flight system.
Matrix-assisted laser ionization and desorption time-of-flight mass spectrometry is a recently developed technique which is particularly useful for the sensitive analysis of large biomolecules. The matrix is a material that assists in the transfer of energy to the analyte molecule, allowing it to be ionized without significant fragmentation, and leave the surface of a target that is being irradiated with a laser. Typically, a few microliters of a solution containing sample molecules at concentrations of about 1 xcexcg/xcexcL are mixed with 10-20 xcexcL of a solution containing matrix molecules at concentrations of about 10 xcexcg/xcexcL. A few microliters of this mixture are then deposited on a suitable substrate and dried in air.
As the sample dries, crystals of matrix are formed and the sample is thought to be incorporated into the crystal. The substrate is then introduced through a vacuum lock into a time-of-flight mass spectrometer system. In such systems, a high voltage source (often 30 KV or more) will be connected to the substrate.
Once the sample has been introduced into the mass spectrometer, a pulsed laser is used to irradiate the sample on the substrate. The interaction of the laser radiation with the matrix molecules leads, by a process that is only partly understood today, to the formation and desorption of largely intact, ionized sample molecules. Predominantly these ions are of a type known as (M+H)+ ions, that is, the neutral sample molecule (M) is ionized by the attachment of a proton. This ionization process has some similarity to the process called xe2x80x9cchemical ionizationxe2x80x9d used conventionally in gas chromatography/mass spectrometry.
Most frequently these ions are analyzed in so-called linear time-of-flight (TOF) mass spectrometers. The ions, once formed, are accelerated by an electric field and then allowed to travel in straight lines until they are detected. The transit time between ion formation and detection can be used to determine the mass of the species from which the ions are generated. Typical linear TOF systems are described in U.S. Pat. No. 5,045,694 (Beavis and Chait). Such linear devices provide only modest mass resolving power, e.g. 50-800, because they are unable to compensate for various known aberrations. A dominant aberration in such linear systems stems from the fact that the ions are formed with a wide distribution of initial velocities. This means that for an ion of a given mass there will be a distribution of arrival times at the detector that will limit the mass resolving power of such a device, since ions with more initial velocity in the forward direction will arrive sooner than ions with less initial velocity.
Techniques for compensating for such aberrations resulting from the initial velocity distribution in TOF mass spectrometers are well-known. The primary technique is to provide an electrostatic mirror, called a reflectron, which reverses the direction of travel of the ions in such a way that the effects of these initial velocity distributions on ion transit times are eliminated. A recent review article describing such devices is xe2x80x9cTime-of-flight Mass Spectrometry: An increasing Role in the Life Sciencesxe2x80x9d, R. J. Cotter, Biomed. Env. Mass Spectrom., 18: 513-532 (1989). The practice of matrix-assisted laser desorption and ionization in reflectron-based instruments is also known and typically produces mass resolutions of 2000-4000 for molecules less than 5000 Daltons in molecular weight.
In both linear and reflectron-based TOF instruments, it is thought that a significant factor limiting mass resolution is the interaction of sample ions with other desorbed matrix ions and molecules i.e., as the desorbed biomolecules leave the surface of the target, they may encounter a plume of matrix ions and molecules. Interactions with this plume may change the energy of desorbed biomolecule ions, but not in a homogeneous manner. Some biomolecules may gain more kinetic energy, some may lose kinetic energy. Thus, the time of arrival of the biomolecules is not exactly the same because some are flying faster, and some slower, than the mean. The net result is band broadening and a concomitant loss in mass resolution. To date, the inventors are not aware of efforts to narrow the initial velocity distribution by manipulation of the matrix. Crystal formation occurs as the sample/matrix mixture dries down in a largely uncontrolled manner. It is postulated that this leads to variability in the analysis of the sample. In addition, the necessity of mixing sample with matrix prior to dry down often results in inefficient use of sample. Accordingly, it would be desirable to provide methods and apparatus for providing greater consistency in the sample preparation process and more efficient use of sample.
Hillenkamp et al., in British Patent Nos. GB 2236185A, and GB 2236186A, disclose surfaces and matrices for laser desorption of biomolecules from surfaces. GB 2236185A discloses a two-dimensional layer comprising a matrix (xe2x80x9cabsorbing componentxe2x80x9d) underlying the substrate. The application is aimed at macromolecular blotting. GB 2236186A discloses desorption of biomolecules using 337 nm or higher laser radiation on a similar surface. Sinapinic acid is shown as the matrix.
Cottrell, PCT/GB90100973, discloses a method for preparing a sample for analysis by LDMS that includes electrospraying the matrix (nicotinic acid) onto a target surface, then sample in TFA is applied and dried down. Finally, the sample is introduced into the mass spectrometer and a laser is directed onto the sample, desorbing the sample.
Cottrell, PCT/GB90100974, discloses a method for preparing a sample for analysis by LDMS that includes electrospraying a substrate (nitrocellulose) onto a target, depositing a sample dissolved in aqueous 0.1% trifluoroacetic acid (TFA), and then drying it down. Matrix material (nicotinic acid, 30 mM in acetone) is then applied in droplet form to cover the dried-down sample and dissolve the substrate nitrocellulose. Sample, substrate and matrix dry down together in an intermixed form. If the sample is a protein, the protein adsorbs to the nitrocellulose by hydrophobic interactions. Loss of mass resolution is caused when excess matrix is evaporated from the surface of the target, causing the plasma effect described supra.
Cottrell, PCT/GB90100975, additionally discloses the use of various matrix materials such as cinnamic acid, benzoic acid, or coumarin in the methods disclosed above.
Beavis, J. Phys. D: Appl. Phys., 26(3), 442-7, has emphasized the desirability of crystal formation.
Hutchens (Proceedings of the 41 st American Society of Mass Spectrometry Conference on Mass Spectrometry and Allied Topics, May 31-Jun. 4, 1993, pp. 781a-781b) has described a technique called Surface-Enhanced Neat Desorption (SEND), in which energy-absorbing molecules are covalently bonded to an inert substrate, allegedly providing a matrix-free method for introducing large molecular weight biopolymers into the gas phase without fragmentation. However, this technique appears to have limited reproducibility.
Other efforts to laser desorb biomolecules include those of Tanaka et al. (Rapid Commun. Mass Spectrom. 2, 151-153, (1988)) who describe a system for matrix-assisted laser desorption and ionization in which the sample is dissolved in glycerol containing small cobalt particles. Cornett et al, Anal. Chem. 65: 2608-2613 (1993) has described a system in which various energy absorbing molecules such as rhodamine 6G are dissolved along with the sample molecules in a liquid matrix such as 3-nitrobenzyl alcohol.
Williams in U.S. Pat. No. 5,135,870 and Becker in U.S. Pat. No. 4,920,264 describe systems involving frozen layers of ice for the desorption and ionization of DNA. None of these systems have demonstrated surprising resolution to date.
Thus the need exists for a laser desorpt on matrix composition that can decrease the loss in mass resolution that occurs when using prior art procedures as well as provide increased sensitivity.
The present invention overcomes the disadvantages and limitations of the prior art by providing a surprising enhancement in mass resolution comprising a thin-layer for sample analysis by laser desorption mass spectrometry wherein the band-broadening contribution due to the matrix substrate sample composition is minimized. In accordance with the invention, mass resolution and sensitivity are increased by the use of a thin layer comprising a matrix material in a supported dispersion wherein the support is a solid or is formed from a solid. The solid support is preferably a solid that limits matrix crystal growth, most preferably a polymer. Thicknesses not greater than 1 xcexcm are most preferred.
The preferred embodiment of the invention is a thin layer for sample analysis by matrix assisted laser desorption mass spectrometry, comprising a matrix-polymer composition disposed upon a substrate. Any matrix material may be used, but Sinapinic acid or xcex1-cyano-4 hydroxycinnamic acid is preferred. The matrix-polymer composition may include a polymer selected from the group consisting of cellulose acetate, cellulose nitrate, and polycarbonate. However, other polymers are also possible. The matrix-polymer composition may preferably range from about 70% polymer to about 30% polymer. Preferably, it is 50%. The mass resolution of the sample is significantly enhanced using this invention, and unexpected mass resolutions of over 8000 have been demonstrated in a linear 150-cm flight tube machine.
The invention is also directed to a device for performning matrix-assisted laser desorption mass spectrometry of sample molecules, comprising a substrate capable of receiving on its surface a thin layer as previously described. The device is combined with a solution of sample molecules wherein the sample and matrix are substantially coplanar, and then subjected to mass analysis.
The substrate underlying the device is selected from the group consisting of glass, ceramic, plastic, metal, or similar materials. The thin layer of the device is resistant to decreased mass resolution and sensitivity over time, i.e., it has a substantial shelf life.
The invention is also directed to a method for making a thin layer for sample analysis by matrix-assisted laser desorption mass spectrometry, the thin layer comprising a matrix material in a supported dispersion wherein the support is a solid, comprising the steps of: depositing a solution containing matrix, support and solvent upon a substrate; and then evaporating the solvent, thereby interspersing the matrix and support on the substrate in a thin layer. The preferred method is spin casting, however other methods are also possible. The product made by this process is also a part of this invention.